DESCRIPTION: Despite exciting recent progress in ex vivo lung bioengineering utilizing decellularized whole lung scaffolds, a number of hurdles remain. These include schemes for functional recellularization with combinations of appropriate cell types, long term maintenance of recellularizing lungs, and incomplete understanding of optimal vascular perfusion and cyclic mechanical stretch influences on proliferation, differentiation, and appropriate function of cells inoculated into the scaffolds. Further, most progress to date has been made in rodent models. This in part reflects the relative difficulties in working with human as compared to rodent lungs including a more limited supply of human lungs and the practical aspects of handling larger more cumbersome lungs. This has hampered progress in assessing the multiple combinatorial conditions that must be evaluated for development of functional human lung tissue. To address these hurdles, we have developed novel and innovative techniques for studying recellularization of acellular human lung scaffolds. These include; a) More optimal detergent- based decellularization protocols; b) Infrared perfusion imaging and sophisticated mass spectrometric assessment of residual scaffold proteins; and c) Novel high throughput approaches for studying decellularized human lungs. This latter technique involves dissection of multiple small 1cm3 segments, each with a cannulatable bronchovascular bundle, from whole decellularized lungs. We have further developed a flexible artificial pleural coating for use with these segments that allows study of ventilation and perfusion of the recellularizing human lung segments. We have also developed a functional assay for surfactant production by assessing changes in lung mechanics in recellularizing scaffolds. Using these techniques, we have made significant advances in recellularizing acellular scaffolds produced from normal and diseased rodent, pig, primate, and human lungs. In particular, we have found significant effects of perfusion and of cyclic mechanical stretch on survival and differentiation, respectively, of pulmonary vascular endothelial cells and of type 2 alveolar epithelial cells in the scaffolds. The goal of the current application is to continue to develop advanced translational applicable schemes for recellularization of human lung scaffolds. Focus will be on combinations of relevant human lung cell types, including endogenous airway progenitor cells and iPS-derived lung epithelial cells (Specific Aim 1), developing appropriate perfusion schemes and perfusates for long term maintenance or recellularizing scaffolds (Specific Aim 2), and developing optimal mechanical ventilation schemes that will maximize influence of cyclic mechanical stretch on development of functional lung tissue (Specific Aim 3). Importantly, we have built an outstanding collaborative team with which to achieve these goals.